beverages
Article
Why Craft Brewers Should Be Advised to Use Bottle
Refermentation to Improve Late-Hopped
Beer Stability
Carlos Silva Ferreira , Etienne Bodart and Sonia Collin *
Earth and Life Institute ELIM, Université catholique de Louvain, Croix du Sud, 2 box L7.05.07,
B-1348 Louvain-la-Neuve, Belgium; carlos.silva@uclouvain.be (C.S.F.); etienne.bodart@uclouvain.be (E.B.)
* Correspondence: sonia.collin@uclouvain.be; Tel.: +32-10472913
Received: 28 February 2019; Accepted: 13 May 2019; Published: 4 June 2019
Abstract: The aromatic complexity of craft beers, together with some particular practices (use of
small vessels, dry hopping, etc.), can cause more oxidation associated with pre-maturated colloidal
instability, Madeira off-flavors, bitterness decrease, and aroma loss. As bottle refermentation is widely
used in Belgian craft beers, the aim of the present work is to assess how this practice might impact their
flavor. In fresh beers, key flavors were evidenced by four complementary techniques: short-chain
fatty acids determination, esters analysis, XAD-2 extract olfactometry, and overall sensory analysis.
In almost all of the fresh beers, isovaleric acid was the sole fatty acid found above its sensory threshold.
Selected samples were further analyzed through natural aging at 20 ◦ C. The presence of yeast in the
bottle minimized the trans-2-nonenal released from Schiff bases and proved less deleterious than
suggested by previous studies with regard to fatty acid release and ester decrease through aging.
Furthermore, according to the yeast species selected, some interesting terpenols and phenols were
produced from glucosides during storage.
Keywords: craft beer; bottle refermentation; AEDA; short-chain fatty acids; beer aging
1. Introduction
Worldwide, over the last few decades, the production of craft beers has grown significantly,
with new commercial products launched onto the market every day. Craftsmen can bring distinctive
flavors to their beers by working with special malts, dual-purpose hop varieties (with or without
dry hopping), spices, and/or specialty yeasts. These are known to impart fruity esters [1,2] and, in
some cases, typical phenolic flavors [3] (e.g., 4-vinylguaiacol brought by phenolic off-flavor (POF(+))
yeasts). In addition, odorous heterocyclic compounds can be issued from colored malts [4,5], while hop
terpenols and polyfunctional thiols bring pleasant citrus and exotic flavors to late- and dry-hopped
beers [6–8]. Unfortunately, the use of small vessels and craftsmanship, by definition, lead to a higher
risk of oxidation and shelf-life decrease.
Beer aging has been the focus of much interest for decades, with the development of worldwide
beverage exchanges. The Dalgliesh plot [9] describes aromatic changes occurring in lager beers
during storage. A linear decrease in bitterness (degradation of isohumulones and/or humulinones)
coincides with an increase in sweet aroma and toffee flavor, together with the well-known cardboard
taint (caused by trans-2-nonenal) [10–13] and ribes off-flavor (a catty smell linked to the presence of
3-sulfanyl-3-methylbutyl formate) [14,15]. Aging of specialty beers is even more complex, with defects
such as Madeira off-flavor [16], phenolic perception [17], a change in hoppy aromas [18], and a detected
ether taint [19].
Bottle refermentation has been widely used by Belgian craft brewers for its carbonation effect,
giving beer the desired effervescence, and also for the associated oxygen consumption, which limits
Beverages 2019, 5, 39; doi:10.3390/beverages5020039
www.mdpi.com/journal/beverages
Beverages 2019, 5, 39
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oxidation and the development of related off-flavors [20–22]. About half a million yeast cells per mL
are pitched into the beer before bottling, in the presence of added fermentable sugars. The beer is then
kept in a warm room (20–28 ◦ C) from two to four weeks.
According to Saison et al. [23], however, refermentation can be damageable, causing loss of
flowery, fruity, and ester notes that are highly appreciated by consumers. Long storage can lead to
yeast autolysis with release of esterases (deleterious to fruity esters) and to excretion of amino acids,
peptides, and short-chain fatty acids [24–28]. When Brettanomyces strains are present in the bottle,
production of isovaleric, hexanoic, and octanoic acids is especially promoted [29–31].
The aim of the present paper was to assess how bottle refermentation impacts the flavor
properties of Belgian craft beers. As bottle refermentation was already known to significantly
improve the release of free-hop thiols from cysteine and glutathione conjugates [22], we decided to
investigate only non-dry-hopped commercial samples. First, short-chain fatty acids were investigated
in 16 bottle-refermented and two unrefermented Belgian craft beers to determine whether they were
present above their sensory threshold. In a few selected samples, more flavors were then analyzed
through natural aging at 20 ◦ C in the dark. Esters (isoamyl acetate, ethyl hexanoate, and ethyl octanoate)
were quantitated by headspace-GC-FID, and most trace aromas were monitored by GC-olfactometry
after XAD-2 aroma extraction. Lastly, some cardboard defects (trans-2-nonenal) and a few other
changes in aroma were evidenced by overall sensory analysis.
2. Materials and Methods
2.1. Materials
Isoamyl acetate (99%), ethyl hexanoate (99%), ethyl octanoate (99%), 2-pentanol (98%), isovaleric
acid (99%), hexanoic acid (≥98%), octanoic acid (≥98%), nonanoic acid (99%), and decanoic acid
were purchased from Sigma Aldrich GmbH (Bornem, Belgium); n-hexanol from Acros Organics
(Geel, Belgium); ethanol (99.8%) from Merck (Darmstadt, Germany); XAD-2 resin from Supelco Inc.
(Bellefonte, United States of America); and sodium chloride, copper sulfate (II), and diethyl ether from
VWR International (Leuven, Belgium). Authentic standard flavor compounds for olfactometry were of
pure grade (purity >98%) from Sigma-Aldrich. Milli-Q water was used (Millipore, Bedford, MA, USA).
2.2. Beer Samples and Aging Procedure
A total of 18 commercial, top-fermented, late-hopped beers (here listed as A–R for reasons of
confidentiality) were kindly supplied by Belgian craft brewers. All were bottle-refermented, except
beers A and B. Six representative samples (A–F), same lot as above, were further selected for more
in-depth investigations through natural aging (20 ◦ C in the dark). The main characteristics of these
beers are depicted in Table 1.
Table 1. Main characteristics of the six selected craft beers.
Beer
Alcohol
(% vol)
Real Extract
(◦ P)
pH
Bitterness
(BU)
Color
(EBC)
Sensorial Characteristics
A
B
C*
D*
E*
F*
6.5
12.3
7.9
8.8
8.1
7.5
4.6
6.6
5.1
5.8
4.2
3.7
4.2
4.4
4.5
4.4
4.4
4.5
15
20
21
14
29
24
12.5
25
16.5
66
14.5
15.5
Butter, apple, hop, green
Alcohol, banana, cheese, phenols
Butter, sulfur, hop
Malt, sulfur, green
Lemon, banana, apple, spicy, phenols
Orange, pineapple, spicy, phenols
*: with bottle refermentation.
Beverages 2019, 5, 39
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2.3. Short-chain Fatty Acid Analysis
First, 100 µL of internal standard (IST—1000 mg/L nonanoic acid) was added to 10 mL of beer
in a 20-mL vial flask, which was immediately closed and shaken for 10 s. Then, 300 µL of n-hexanol
was added before shaking again for 5 min. Compounds were recovered in assembled n-hexanol
fractions after 2 successive centrifugations (14,000 rpm) [32]. Next, 1 µL of extract was analyzed on an
Agilent 6890N gas chromatograph equipped with a split injector maintained at 200 ◦ C (split ratio = 73.6).
The FID (flame ionization detector) was set at 220 ◦ C. Compounds were injected into a CP-Wax 58 column
(Agilent, 60 m × 0.32 mm i.d., 0.5-µm film thickness). The carrier gas was nitrogen, and the pressure was
set at 60 kPa. The oven temperature was programmed to rise from 125 to 140 ◦ C at 8 ◦ C/min and then to
180 ◦ C at 15 ◦ C/min. Quantitation was done by determining the relative response of each compound to
IST (done by standard addition to beer B). Results are expressed as the average of duplicates.
2.4. Static Headspace Analysis of Esters
Prior to analysis, the beers were stored for 2 h at 4 ◦ C to avoid excessive foaming. The whole
procedure was carried out in a cold room (4 ◦ C). Then, 40 µL of internal standard (IST, 2500 mg/L
2-pentanol) and 1.9 g of sodium chloride were added to 5 mL of beer in a 20-mL screw vial flask, which
was closed immediately and kept closed until analysis. A total of 500 µL of extract were analyzed on a
Thermo Finnigan Trace gas chromatograph, equipped with a splitless injector maintained at 250 ◦ C;
the split vent was opened 1 min post-injection. The FID detector was set at 260 ◦ C. Compounds were
injected into a VF-Wax MS column (Agilent, 60 m × 0.32 mm i.d., 0.5-µm film thickness). The carrier
gas was nitrogen, and the pressure was set at 100 kPa. The oven temperature was programmed to
rise from 40 to 140 ◦ C at 8 ◦ C/min and then to 180 ◦ C at 15 ◦ C/min. Quantitation was performed by
standard addition (relative response of each compound to IST). Results are expressed as the average
of duplicates.
2.5. Flavor XAD-2 Extraction and Gas Chromatography—Olfactometry Analytical Conditions
An extraction procedure based on that of Lermusieau et al. [33], was used to recover aroma
compounds from beer. First, 4 g of XAD-2 resin were added to 50 mL of beer in a 250-mL flask.
The flask was sealed with a Teflon-lined cap and shaken in the dark for 2 h at 200 rpm. After extraction,
the contents were poured into a glass column with a coarse frit and Teflon stopcock, and the liquid
was drained off, leaving a small bed of resin, which was further rinsed with 100 mL of distilled
water (4 × 25 mL). Aroma compounds were then eluted with 40 mL of diethyl ether (2 × 20 mL).
The extract was dried with Na2 SO4 and concentrated to 0.5 mL in a Kuderna-Danish evaporator at
39 ◦ C. A Chrompack CP9001 gas chromatograph equipped with a splitless injector maintained at 250 ◦ C
was used for the olfactometry analyses, and the split vent was opened after 0.5 min. Compounds were
separated using a wall-coated open-tubular (WCOT) apolar CP SIL5 CB capillary column (Agilent,
50 m × 0.32 mm, 1.2-µm film thickness) connected to a GC-odor port at 250 ◦ C. The eluent was diluted
with a large volume of air (20 mL/min) previously humidified using aqueous copper (II) sulfate solution.
The oven temperature was programmed from 36 to 85 ◦ C at 20 ◦ C/min, to 145 ◦ C at 1 ◦ C/min, to 250 ◦ C
at 3 ◦ C/min, and then to remain constant at 250 ◦ C for 30 min. A volume of 1 µL of extract was injected.
Sniffing was performed by two experienced panelists. Serial dilutions were prepared from the initial
XAD-2 extract at a ratio of 1:3n in diethyl ether. The dilution factor (FD) was calculated as 3n , n + 1
being the number of dilutions (factor 3) required for no odor to be perceived (Log3 FD values in Table 2
equal to 0, 1, 2, . . ., 10, nd if no odor detected for the undiluted extract). The difference between two
Log3 FD becomes significant when above 1.
Beverages 2019, 5, 39
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Table 2. GC-olfactometric analysis of XAD-2 extracts issued from beers A, C, E, and F (fresh and after 6
months of storage at 20 ◦ C). RI: retention index; nd: not detected.
Log3 FD
A
C
E
F
Compound
RI
Odor
Fresh
Aged
Fresh
Aged
Fresh
Aged
Fresh
Aged
Ethyl butyrate
778
Red fruits
1
1
4
6
nd
3
3
4
3-Methyl-2buten-1-thiol
809
Garlic, hoppy
7
8
7
8
10
10
10
10
Isovaleric acid
811
Sweat, rancid
2
2
2
2
4
5
5
5
2-Methylbutanoic
acid
828
Sweat
2
2
2
2
4
5
3
6
2-Methyl-3furanthiol
850
Broth
5
7
5
2
10
10
10
10
Isoamyl acetate
854
Fruity, banana
1
1
2
2
nd
nd
nd
nd
Ethyl hexanoate
979
Fruity, candy
2
2
4
4
3
4
4
4
Furaneol
1037
Cotton candy
4
5
6
7
5
5
4
7
Linalool
1089
Flowery,
coriander
7
6
6
7
nd
nd
5
2
trans-2-Nonenal
1127
Cardboard
nd
6
nd
5
3
4
4
5
Citronellol
1216
Fruity, flowery
nd
nd
1
4
3
2
nd
8
4-Vinylguaiacol
1294
Clove
3
4
4
4
5
6
4
6
γ-Nonalactone
1327
Coconut
nd
3
0
4
1
2
3
5
Vanillin
1365
Vanilla
0
nd
6
7
nd
4
1
5
β-Damascenone
1374
Fruity, apricot
2
3
4
5
4
4
3
6
4-Vinylsyringol
1543
Clove
3
3
0
3
2
3
5
5
2.6. Sensory Analyses
A group of 10 panelists (all trained scientists, non-smokers, and regular consumers of craft beers,
including three women and seven men aged 23–55 years) scored four aging attributes on a scale of
0–4: cardboard, bread, cooked fruit, and dried fruit. A score of 0 meant the panelist did not detect the
aroma, whereas a score of 4 meant the aroma was strongly perceived.
3. Results and Discussion
3.1. Short-Chain Fatty Acids
GC analyses revealed considerable variability of short-chain fatty acid profiles among fresh
Belgian craft beers (Figure 1). Most samples contained isovaleric acid (Figure 1a) at a concentration
above its threshold (1 mg/L) [34]. In all cases, on the other hand, hexanoic (Figure 1b), octanoic
(Figure 1c), and decanoic acids (Figure 1d) were below their sensory thresholds (10, 10, and 5 mg/L,
respectively [35]). A good correlation (R2 = 0.72) was found, as expected, between the concentrations
of hexanoic acid and octanoic acid (Figure 2).
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Octanoic
acid
(mg/L)
Octanoic
acid
(mg/L)
Figure 1. Concentrations of isovaleric (a), hexanoic (b), octanoic (c), and decanoic (d) acids (mg/L) in
Figure
1.Belgian
Concentrations
of The
isovaleric
(a),
hexanoic
(b),
octanoic
(c),the
and
decanoic (d)
(d)
acidsthreshold
(mg/L) in
in
18
fresh1.
craft beers.
dotted(a),
lines
in each(b),
graph
indicate
compound
sensory
Figure
Concentrations
of
isovaleric
hexanoic
octanoic
(c),
and
decanoic
acids
(mg/L)
18fresh
freshBelgian
Belgiancraft
craft
beers.
The
dotted
lines
in each
graph
indicate
the compound
sensory
threshold
(Thr.).
18
beers.
The
dotted
lines
in each
graph
indicate
the compound
sensory
threshold
(Thr.).
(Thr.).
12
12
10
10
8
8
6
6
4
4
2
2
0
0 0
0
R² = 0.7281
R² = 0.7281
2
4
6
Hexanoic
acid
(mg/L)
2
4
6
Hexanoic acid (mg/L)
Figure 2. Correlation between the concentrations (mg/L) of octanoic acid and hexanoic acid (in
Figure 2. Correlation between the concentrations (mg/L) of octanoic acid and hexanoic acid (in eighteen
Figure 2.fresh
Correlation
between the concentrations (mg/L) of octanoic acid and hexanoic acid (in
eighteen
beers: A–R).
fresh beers: A–R).
eighteen fresh beers: A–R).
In
acids
were
determined
after
3, 6,
12
In aa representative
representativeset
setofofbeers
beers(A–F),
(A–F),short-chain
short-chainfatty
fatty
acids
were
determined
after
3, and
6, and
In aofrepresentative
set
of beers
(A–F),
short-chain
fatty acids
were determined
after
3, 6, and
12
months
natural
aging
(Figure
3).
No
increase
in
isovaleric,
hexanoic,
octanoic,
or
decanoic
acid
12 months of natural aging (Figure 3). No increase in isovaleric, hexanoic, octanoic, or decanoic acid
months of natural observed
aging (Figure 3).
No increase
in isovaleric,
hexanoic, octanoic, isorthe
decanoic acid
concentration
concentration was
was observed over
over the
the aging
aging period.
period. Worth
Worth mentioning,
mentioning, however,
however, is the significant
significant
concentration
was
observed
over
the
aging
period.
Worth
mentioning,
however,
is
the
significant
decrease
in
octanoic
acid
in
beer
C
(Figure
3C).
This
was
the
only
beer
tested
here
in
decrease in octanoic acid in beer C (Figure 3C). This was the only beer tested here in which
which the
the
decrease in octanoic
acid in beer C (Figure
3C).before
This was
theInonly
beer tested here
in which the
concentration
aging.
concentration of
of this
this compound
compound reached
reached 99 mg/L
mg/L before
aging.
In conclusion,
conclusion, bottle
bottle refermentation
refermentation
concentration
of this compound
reached
9 mg/L beforenatural
aging. In conclusion,
bottle refermentation
does
does not
not cause
cause significant
significant release
release of
of fatty
fatty acids
acids through
through natural aging
aging and
and thus
thus does
does not
not negatively
negatively
does
not
cause
significant
release
of
fatty
acids
through
natural
aging
and
thus
does not negatively
impact
flavor.
impact flavor.
impact flavor.
Beverages 2019, 5, 39
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Unrefermented
Isovaleric acid
Hexanoic acid
Octanoic acid
Decanoic acid
A
8
6
4
2
10
Concentration (mg/L)
Concentration (mg/L)
10
B
8
6
4
2
0
0
Fresh
3 months
Fresh
6 months 12 months
3 months
6 months
12 months
Bottle-refermented
10
C
Concentration (mg/L)
Concentration (mg/L)
10
8
6
4
2
6
4
2
0
0
Fresh
3 months
6 months
12 months
Fresh
3 months
Fresh
3 months
6 months
12 months
10
10
E
Concentration (mg/L)
Concentration (mg/L)
D
8
8
6
4
2
0
F
8
6
4
2
0
Fresh
3 months
6 months
12 months
6 months
12 months
Figure 3. Concentrations (mg/L) of isovaleric acid (▲), hexanoic acid (■), octanoic acid (●), and
Figure 3. Concentrations (mg/L) of isovaleric acid (N), hexanoic acid (), octanoic acid (), and decanoic
decanoic acid (♦) in fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months).
acid () in fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months). Variation
Variation coefficients under 5%.
coefficients under 5%.
3.2.Esters
Esters
3.2.
Esterswere
weredetermined
determinedon
onthe
thesame
samesampling
samplingofofcraft
craftbeers
beers(A–F)
(A–F)ininthe
thecourse
courseofofone
oneyear
yearofof
Esters
2=
2 = 0.86) was again observed between
naturalaging
aging(Figure
(Figure4).4).For
Forfresh
freshbeers,
beers,a astrong
strongcorrelation
correlation(R(R
natural
0.86) was again observed between
theconcentrations
concentrations
ethyl
hexanoate
and
ethyl
octanoate
(Figure
although
did
the
ofof
ethyl
hexanoate
and
ethyl
octanoate
(Figure
5a), 5a),
although
thesethese
esterester
levelslevels
did not
not correlate
well the
withhexanoic
the hexanoic
and octanoic
concentrations
(Figure
hexanoate
correlate
well with
and octanoic
acids acids
concentrations
(Figure
5b,c). 5b,c).
Ethyl Ethyl
hexanoate
and
and ethyl
octanoate,
were found
very to
close
tosensory
their sensory
thresholds
(0.20.9
and
0.9 mg/L,
respectively
ethyl
octanoate,
were found
very close
their
thresholds
(0.2 and
mg/L,
respectively
[36])
in allsamples
fresh samples
and remained
relatively
stableaging,
during
aging,
with no significant
in[36])
all fresh
and remained
relatively
stable during
with
no significant
differencedifference
between
between
the unrefermented
and
B) and bottle-refermented
(C–F) samples.
On the
otherthe
hand,
the
the
unrefermented
(A and B)(A
and
bottle-refermented
(C–F) samples.
On the other
hand,
fruity
fruity banana-like
wasto
found
to be partially
degraded
throughout
the
of storage
banana-like
isoamylisoamyl
acetate acetate
was found
be partially
degraded
throughout
the year
ofyear
storage
in all
in allexcept
beersBexcept
B (characterized
byhigher
a much
higher
level of
ethanol,
1). trend
The similar
trend
beers
(characterized
by a much
level
of ethanol,
Table
1). TheTable
similar
observed
in
A and C–F
esters can
be broken
down even
without
releaseupon
of esterases
Aobserved
and C–F in
confirms
thatconfirms
esters canthat
be broken
down
even without
the release
ofthe
esterases
yeast
upon yeast
In all of
the
beers, the
isoamyl
acetate concentration
remained
above the
sensory
autolysis.
In autolysis.
all of the beers,
the
isoamyl
acetate
concentration
remained above
the sensory
threshold
threshold
(0.5after
mg/Lone
[37])
after one year.
(0.5
mg/L [37])
year.
Beverages 2019, 5, 39
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Concentration (mg/L)
6.0
Isoamyl acetate
Ethyl hexanoate
Ethyl octanoate
A
4.5
3.0
1.5
0.0
Concentration (mg/L)
Unrefermented
6.0
B
4.5
3.0
1.5
0.0
Fresh
3 months 6 months 12 months
Fresh
3 months 6 months 12 months
Bottle-refermented
6.0
C
Concentration (mg/L)
Concentration (mg/L)
6.0
4.5
3.0
1.5
Fresh
3.0
1.5
3 months 6 months 12 months
6.0
E
Concentration (mg/L)
Concentration (mg/L)
4.5
0.0
0.0
6.0
D
4.5
3.0
1.5
0.0
Fresh
3 months 6 months 12 months
Fresh
3 months 6 months 12 months
F
4.5
3.0
1.5
0.0
Fresh
3 months 6 months 12 months
Figure 4. Concentrations (mg/L) of isoamyl acetate (▲), ethyl hexanoate (■), and ethyl octanoate (●)
Figure 4. Concentrations (mg/L) of isoamyl acetate (N), ethyl hexanoate (), and ethyl octanoate () in
in fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months). Variation
fresh beers (A–F) and their evolution during natural aging (3, 6, and 12 months). Variation coefficients
coefficients under 5%.
under 5%.
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0.8
(a)
Ethyl hexnaoate (mg/L)
Ethyl octanoate (mg/L)
1.2
8 of 11
1.0
0.8
0.6
0.4
0.2
R² = 0.8665
0.0
0.0
0.2
0.4
0.6
Ethyl hexanoate (mg/L)
Ethyl octanoate (mg/L)
1.2
0.8
(b)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
R² = 0.2476
0.0
0
2
4
Hexanoic acid (mg/L)
6
(c)
1.0
0.8
0.6
0.4
0.2
R² = 0.0985
0.0
0
2
4
6
8
Octanoic acid (mg/L)
10
Figure 5.
5. Correlations
(mg/L)
(a)(a)
ethyl
octanoate
andand
ethyl
hexanoate,
(b)
Figure
Correlationsbetween
betweenthe
theconcentrations
concentrations
(mg/L)
ethyl
octanoate
ethyl
hexanoate,
ethyl
hexanoate
and
hexanoic
acid,
and
(c)
ethyl
octanoate
and
octanoic
acid
(in
six
fresh
beers:
A–F).
(b) ethyl hexanoate and hexanoic acid, and (c) ethyl octanoate and octanoic acid (in six fresh beers: A–F).
3.3. Olfactive Analysis of XAD-2 Extracted Flavors
Samples A,
A,C,C,E,E,
and
F blond
(all blond
to the
avoid
the complexity
special malt-derived
and
F (all
beersbeers
to avoid
complexity
of specialofmalt-derived
molecules
molecules
largely investigated
[5,17])
were subjected
XAD-2
resin extraction
largely investigated
elsewhere elsewhere
[5,17]) were
subjected
to XAD-2toresin
extraction
followedfollowed
by GCby
GC-olfactometric
analysis.
Beer
odor intensities
were determined
by theextract
aromadilution
extract
olfactometric
(GC-O)(GC-O)
analysis.
Beer odor
intensities
were determined
by the aroma
dilution
To focus
on beer
flavor-active
compounds,
Table22 only
only those
analysis analysis
(AEDA)(AEDA)
[38]. To[38].
focus
on beer
flavor-active
compounds,
we we
listlist
in inTable
compounds whose FD was as high as that of ethyl
ethyl hexanoate,
hexanoate, an ester known to be present in the
the
samples at concentrations
concentrations close
close to
to its
its sensory
sensory threshold.
threshold.
3-Methyl-2-buten-1-thiol
3-Methyl-2-buten-1-thiol(Log
(Log33FD
FD== 7–10, a pleasant hoppy flavor here but also known as skunky
at aa much
much higher
higher level
level[22]),
[22]),2-methyl-3-furanthiol
2-methyl-3-furanthiol(Log
(Log33FD
FD == 2–10, broth),
broth), furaneol
furaneol (Log
(Log33FD =
= 4–7,
cotton
FD
=
2–7,
flowery/coriander)
emerged
as
the
most
potent
odorants
in
cotton candy),
candy),and
andlinalool
linalool(Log
(Log
3
FD
=
2–7,
flowery/coriander)
emerged
as
the
most
potent
odorants
3
all
four
beers.
TheThe
persistent
detection
of 4-vinylguaiacol
(Log(Log
3–6)
in allinsamples
indicates
that
in all
four
beers.
persistent
detection
of 4-vinylguaiacol
FD
= 3–6)
all samples
indicates
3 FD 3=
that POF(+)
strains
bybrewers.
the brewers.
POF(+)
strains
had had
beenbeen
usedused
by the
already mentioned
mentioned above, even in bottle-refermented
bottle-refermented beers (C, E, and
and F),
F), isoamyl
isoamyl acetate
acetate
As already
(Log33FD ==1–2,
in E
F due
thetostrong
previous
odor) and
ethyl
hexanoate
(Log3FD
(Log
1–2,undetectable
undetectable
inand
E and
F to
due
the strong
previous
odor)
and
ethyl hexanoate
= 2–4)
showed
good
stability
through
aging,
with
no
significant
changes
in
FD.
On
the
other
hand,
(Log
FD
=
2–4)
showed
good
stability
through
aging,
with
no
significant
changes
in
FD.
On
the
other
3
the red-fruit
ethyl ethyl
butyrate
was produced
during
storage
in the
beers
(from
not
hand,
the red-fruit
butyrate
was produced
during
storage
inbottle-refermented
the bottle-refermented
beers
(from
detected
to Log
3FD3=
3 in
E and
fromfrom
4 to 46 to
in 6beer
C). C).
not
detected
to Log
FD
= beer
3 in beer
E and
in beer
Although trans-2-nonenal
trans-2-nonenalshowed
showedan
anincrease
increasein
in all
all four
four beers
beers during
during aging,
aging, the
the highest
highest FD jump
Although
was observed
observed for
for the
the unrefermented
unrefermented A
A (from
(fromnot
notdetected
detectedto
toLog
Log33FD
FD =
= 6).
was
interesting result
result was the strong increase, especially in aged beer F,
F, of compounds suspected
An interesting
released during storage through
through glucoside
glucoside hydrolysis:
hydrolysis: citronellol, 4-vinylguaiacol, vanillin, and
to be released
β-damascenone [39,40].
[39,40]. In
Inthis
thiscase,
case,the
theselected
selected
yeast
clearly
brought
flavors
F, explaining
β-damascenone
yeast
clearly
brought
newnew
flavors
to F,to
explaining
why
why some
consumers
prefer
the six-month-aged
efficiency
of the
yeast
β-1,3-glucanase
some
consumers
may may
prefer
the six-month-aged
beer.beer.
TheThe
efficiency
of the
yeast
β-1,3-glucanase
or
or β-glucosidase
should
be taken
into account
to predict
the amounts
inaglycons
which aglycons
can be
β-glucosidase
should
be taken
into account
to predict
the amounts
in which
can be released
released through aging. Linalool (detected in fresh beers A, C, and F) was the sole hop terpenol found
Beverages 2019, 5, 39
Beverages 2019, 4, x FOR PEER REVIEW
9 of 11
9 of 11
through
aging.
Linalool
beers A, C,
andterpenol
F) was biotransformations
the sole hop terpenol
found
to
to decrease
in beer
F (Log3(detected
FD from 5intofresh
2), suggesting
yeast
in the
bottle
decrease
in
beer
F
(Log
FD
from
5
to
2),
suggesting
yeast
terpenol
biotransformations
in
the
bottle
[41].
[41].
3
3.4.
Sensory Analyses
Analyses
3.4. Sensory
Beers
Beers A–F
A–F were
were investigated
investigated by
by aa trained
trained panel
panel while
while fresh
fresh and
and then
then after
after 33 and
and 66 months
months of
of
storage.
As
suspected
from
the
GC-olfactometry
results,
unrefermented
beers
A
and
B
developed
storage. As suspected from the GC-olfactometry results, unrefermented beers A and B developed a
arelatively
relativelyintense
intensecardboard
cardboardflavor
flavor(trans-2-nonenal),
(trans-2-nonenal),already
alreadystrongly
strongly perceived
perceived after
after 33 months
months
(Figure
6).
Despite
the
increase
in
FD
for
furaneol
and
β-damascenone
after
6
months
(Figure 6). Despite the increase in FD for furaneol and β-damascenone after 6 months of
of storage,
storage, the
the
attributes
attributes bread,
bread, dried
dried fruit,
fruit, and
and cooked
cooked fruit
fruit remained
remained absent
absent or
or relatively
relativelyweak.
weak.
A
3
2
F
B
1
0
E
C
D
Fresh
3 months
6 months
Figure 6.6. Spider
Spiderdiagram
diagramofof
cardboard
flavor
intensity
in fresh
(●),naturally
and naturally
and 6
Figure
cardboard
flavor
intensity
in fresh
(), and
aged (3aged
and 6(3
months)
months)
beers
(A–F).
beers (A–F).
4. Conclusions
Bottle refermentation of craft beers can be promoted for its ability both to protect beer against
against
oxidation, (this protection is required to avoid colloidal instability, bitterness
decrease,
and
aroma
bitterness
loss) and to avoid the accumulation of trans-2-nonenal through enzymatic reduction to nonenol and
nonanol.
inin
thethe
bottle
proved
notnot
so deleterious
as itas
regards
fatty fatty
acid
nonanol. Moreover,
Moreover,the
thepresence
presenceofofyeast
yeast
bottle
proved
so deleterious
it regards
excretion
and
ester
hydrolysis
during
the
first
year
of
storage,
while
leading
to
the
release
of
interesting
acid excretion and ester hydrolysis during the first year of storage, while leading to the release of
terpenols
phenolsand
from
aglycons.
interestingand
terpenols
phenols
from aglycons.
Author
Conceptualization, S.C.;
S.C.; Data
Data curation,
curation, C.S.F.,
C.S.F., E.B.,
E.B., and
and S.C.;
S.C.; Formal
Formal analysis,
analysis, C.S.F.,
C.S.F., E.B.,
E.B.,
Author Contributions:
Contributions: Conceptualization,
and S.C.; Writing—original draft, C.S.F. and S.C.
and S.C.; Writing—original draft, C.S.F. and S.C.
Funding: This research received no dedicated funding.
Funding: This research received no dedicated funding.
Conflicts of Interest: The authors declare no conflicts of interest.
Conflicts of Interest: The authors declare no conflicts of interest.
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